The transcription factor dfoxo controls the expression of insulin pathway genes and lipids content under heat stress in Drosophila melanogaster

The insulin/insulin-like growth factor signaling (IIS) pathway is one of the key elements in an organism’s response to unfavourable conditions. The deep homology of this pathway and its evolutionary conservative role in controlling the carbohydrate and lipid metabolism make it possible to use Drosophila melanogaster for studying its functioning. To identify the properties of interaction of two key IIS pathway components under heat stress in D. melanogaster (the forkhead box O transcription factor (dfoxo) and insulin-like peptide 6 (dilp6), which intermediates the dfoxo signal sent from the fat body to the insulin-producing cells of the brain where DILPs1–5 are synthesized), we analysed the expression of the genes dilp6, dfoxo and insulin-like receptor gene (dInR) in females of strains carrying the hypomorphic mutation dilp641and hypofunctional mutation foxoBG01018. We found that neither mutation inf luenced dfoxo expression and its uprise under short-term heat stress, but both of them disrupted the stress response of the dilp6 and dInR genes. To reveal the role of identif ied disruptions in metabolism control and feeding behaviour, we analysed the effect of the dilp641 and foxoBG01018 mutations on total lipids content and capillary feeding intensity in imago under normal conditions and under short-term heat stress. Both mutations caused an increase in these parameters under normal conditions and prevented decrease in total lipids content following heat stress observed in the control strain. In mutants, feeding intensity was increased under normal conditions; and decreased following short-term heat stress in all studied strains for the f irst 24 h of observation, and in dilp641 strain, for 48 h. Thus, we may conclude that dfoxo takes part in regulating the IIS pathway response to heat stress as well as the changes in lipids content caused by heat stress, and this regulation is mediated by dilp6. At the same time, the feeding behaviour of imago might be controlled by dfoxo and dilp6 under normal conditions, but not under heat stress.


Introduction
Nowadays, as living beings often encounter unfavourable en vironmental conditions such as pollution and global warming, the study of deeply conservative mechanisms that contribute to adaptation is of current interest. It is known that such in fluences launch the development of nonspecific adaptive de fensive responses on molecular (Garbuz, Evgen'ev, 2017), behavioral (Kaluev, 1999), biochemical and physiological (Gruntenko, 2008;Even et al., 2012;Miyashita, Adamo, 2020) levels. The ability to respond to stress in an integrated manner, which comprises behavioral, metabolic and molecular reactions, is key for survival and adaptation of animals includ ing insects (Koyama et al., 2020). It was proven that besides its role as crucial modulator of growth and metabolism, in insects, the IIS pathway is an essential component of the neuroendocrine stress reaction Lubawy et al., 2020). Due to the deep homology of this pathway in animals of different taxa including humans and flies, it is possible to use the latter as an object for investigating evolutionaryconservative mechanisms underlying molecu lar genetic regulation of the IIS pathway, and carbohydrate and lipid metabolism it controls. As in most animals, in insects, carbohydrates and lipids serve as the main energy supply (Ar rese, Soulages, 2010). The processes of producing and storing energy undergo complex modulation by many inner factors including heritage, lifestyle, hormones, metabolites, as well as various outside influences (Mattila, Hietakangas, 2017).
Drosophila's applicability to the research of metabolism is defined by the simplicity of its IIS pathway regulation (Fig. 1), which involves homologues of insulin (DILPs1-5) and insulinlike growth factors (DILP6) of mammals connecting to a single insulinlike receptor (dInR), which activates the path way , and two homologues of relaxin (DILPs7,8) (Gontijo, Garelli, 2018). The dInR signal being transduced directly or via its substrate CHICO (the homologue of insulin receptor substrates of mammals, IRS1-4) causes dAkt/PKB (protein kinase В homologue) to activate, which in turn modulates the activity of a number of proteins, in particular, it phosphorylates transcriptional fac tor of Forkhead box class O family, dFOXO (homologue of mammalian FOXO), which is synthesized in the fat body and controls the transcription of more than a thousand genes (Bai et al., 2012), and inhibits its translocation into the nucleus (Puig et al., 2003;Slack et al., 2011;ÁlvarezRendón et al., 2018). Under stress, dFOXO is translocated to the nucleus (Jünger et al., 2003;Hwangbo et al., 2004;Gruntenko et al., 2016) activating the expression of a number of genes including dInR via a feedback loop . It was also previously shown that the expression of dilp6 in the fat body inhibits the expression of dilp2 and dilp5 in imago's brain as well as the secretion of DILP2 into the hemolymph, and that dFOXO influence on the expression of DILPs produced in median neurosecretory cells is mediated by DILP6 synthe sized in the fat body (Slaidina et al., 2009;Bai et al., 2012). Thus, DILPs seems to connect dFOXO, adipose tissue and endocrine function of the brain, creating a feedback loop back to dInR.
Stress reaction causes the mobilization of organism's ener gy reserves along with a variety of metabolic changes. In a changing environment, feeding behaviour plays an important role in adaptation (Rabasa, Dickson, 2016). It is known that in mammals, acute stress is usually accompanied by feeding suppression and a decrease in weight gain; chronic stress can result in excessive food intake, weight gain and obesity (Rabasa, Dickson, 2016).
This study aimed to analyse the expression of dInR, dilp6 and dfoxo genes of three key components of the IIS pathway, which is involved in neuroendocrine stress reaction, in D. melanogaster strains carrying dilp6 41 и foxo BG01018 mutations under heat stress, and to evaluate the latter's influence on feeding behaviour and total lipids content in these strains.

Materials and methods
Drosophila melanogaster strains and stress conditions. Three D. melanogaster strains were used in this study: strain dilp6 41 with the deletion covering the 3′ region of phl gene and 5′ upstream region of dilp6 including the first exon and part of the first intron (Rauschenbach et  foxo BG01018 , which carries a P[GT1] element transposon in the 5′ upstream region of the dfoxo gene, resulting in a mild loss of function (Dionne et al., 2006); and their progenitor strain w 1118 as a control. The stocks were obtained from the Bloomington Drosophila Stock Center (Bloomington, IN, USA). The cultures were raised on standard medium (agaragar, 7 g/l; corn grits, 50 g/l; dry yeast, 18 g/l; sugar, 40 g/l) and kept at 25 °C, 12:12 h photoperiod, relative humidity 50 %. Imagoes were synchronised at eclosion (flies were collected every 3-4 hours). Females were exposed to heat stress by transferring vials with flies from a 25 °C incubator to a 38 °C incubator for 60 or 90 min. After 60 min of stressing flies were returned to 25 °C, after 90 min they were subsequently frozen in liquid nitrogen and stored at -80 °C.
Quantitative real-time polymerase chain reaction (qRT-PCR). mRNA quantity of dilp6, dfoxo and dInR genes was evaluated in whole body homogenates of CantonS fe males (15 flies/sample) using TRI reagent Lot #BCBT8883 (SigmaAldrich, USA) for total RNA extraction, Revert Aid First Strand cDNA Synthesis Kit #K1621 (Thermo Fisher Scientific, USA) with oligo (dT)18 primer for synthesis of cDNA, M427 Kit with SYBRGreen I (Syntol, Russia) and CFX96 Touch qPCR System (BioRad, USA) for performing qRTPCR. Each reaction was performed in triplicates with three biological replicates. Data were normalized against Act5C. High stability of Act5C expression under heat stress was shown by Ponton et al. (2011). The primers used in the study are shown in the Table. Total lipid quantification. Quantification of total lipids was performed using Van Handel's method (1985) modified for D. melanogaster (Eremina, Gruntenko, 2020) under normal conditions or in 24 h after 60 min under 38 °С. Flies (1 fly per sample, 10-20 samples per each studied group) were de capitated to avoid the influence of eye pigment on the mea surement results, homogenised on ice in 100 μl of chloroform methanol (1:1) and shacked for 10 min. 50 μl of supernatant were transferred to new tubes and placed in microthermostat M208 (BisN, Russia) at 90 °С till the solvent completely evaporated. Then 10 μl of 95 % H 2 SO 4 were added to each sample and they were again kept at 90 °С for 2 min. After dFOXO controls the expression of insulin pathway genes and lipids content under stress in Drosophila Each value is a mean of three biological replicates. Error bars show standard error of the mean. Asterisks indicate significant differences between females with mutation of Drosophila insulin-like peptide 6 gene (dilp6 41 ) and females of the control strain w 1118 ( p < 0.001). Diamond indicates significant differences between stressed and control groups of the same genotype ( p < 0.05). Hash indicates a tendency for such differences ( p < 0.07).  Gene mRNA/Act5C mRNA # # ◊ ◊ ◊ * * * that the samples were cooled on ice and the phosphovanillin color reagent (85 % H 3 PO 4 + 6 % vanillin solution (4:1)) was added up to 1 ml of volume. The samples were incubated for 15 min at room temperature till pink colouration appeared and was stable for 1 h. Then the samples were measured by Smart Spec Plus spectrophotometer (BioRad, USA) at 525 nm. Feeding behaviour analysis (CAFE). Ingestion was mea sured using the Capillary Feeder (CAFÉ) method of Ja et al. (2007), modified by Williams et al. (2014). To provide flies with a humid environment, flatbottomed glass vials (20 × 100 mm) with 1 % agarose (5 cm high) were placed into microcentrifuged 50 ml tubes filled with 7 ml of water. Each glass capillary (10 × 90 mm, Narishige, Japan) was filled with 20 μl of liquid food containing 5 % sugar and 5 % east extract (Biospringer, France). Five females were placed into each vial (4-9 vials per group), which was plugged with a foam plug. A capillary was inserted into it through 10 μl and 200 μl pipette tips and was held in place by them. The vials with flies were kept in an incubator (Sanyo, Japan) at 25 °C, 50 % relative humidity, 12:12 h photoperiod for 24 or 48 h. Before that the experimental group was subjected to shortterm heat stress (38 °C, 60 min). Initial and final food levels in capil laries were marked to determine total food consumption per day. To minimize food evaporation, capillaries were topped with a 0.1 μl oil layer. To adjust for food evaporation, a vial without flies was used.
Statistical analysis. Data on gene expression were analyzed by the 2 −∆∆CT method (Livak, Schmittgen, 2001). All data are presented as means ± SEM and analysed by ANOVA. The results were considered significant at p < 0.05.

Results and discussion
To discover whether disruption of the feedback loop of the IIS pathway regulation affects its stress response, we studied the expression of three key genes of the pathway, dilp6, dfoxo and dInR, in D. melanogaster females carrying hypomorphic mutation dilp6 41 and hypofunctional mutation foxo BG01018 under normal conditions or heat stress (38 °С, 90 min). There were no quantitative changes in mRNA expression level of dilp6 and dInR genes in dilp6 41 and foxo BG01018 strains under heat stress, whereas in their progenitor strain w 1118 the expres sion of dilp6 decreased, and the expression of dInR increased under heat stress (Fig. 2, p < 0.05 for both genes). At the same, dfoxo expression level increased or had a tendency to increase under heat stress in all strains under study (see Fig. 2, STRAIN -F (2, 12) = 3.14, p < 0.081; STRESS -F (1, 12) = 12.80, p < 0.0038). Notably, dilp6 41 mutants are characterised by a lower dilp6 expression ( p < 0.001); however, dfoxo expres sion in foxo BG01018 mutants does not differ from the control strain w 1118 (see Fig. 2). This allows us to assume that the previously described loss of dFOXO function in foxo BG01018 strain (Dionne et al., 2006) is connected not with a lowered expression level of the corresponding gene but with a defect in its structure.
The results of qualitative measurement of total lipids in D. melanogaster females with dilp6 41 and foxo BG01018 muta tions under normal conditions or following heat stress (38 °С, 60 min) signify that both mutations cause an increase in lipid content in comparison with the control strain w 1118 , and lipid content in dilp6 41 and foxo BG01018 strains, unlike in their progenitor strain, does not decrease in 24 h after heat stress Each value is a mean of 10-20 (a) and 9-11 (b) measurements. Error bars indicate s.e.m. Asterisk indicates signif icant differences between control females of w 1118 strain and females with dilp6 41 and foxo BG01018 mutations (** p < 0.01, *** p < 0.001). Diamond indicates signif icant differences between stressed and control groups of the same genotype (◊ -p < 0.05, ◊ ◊ -p < 0.01, ◊ ◊ ◊ -p < 0.001).
It was previously shown that the IIS pathway can inter act with gonadotropins and biogenic amines in Drosophila modulating their dynamics under stress and thus participat ing in the control of organism's stress response . However, it remained unclear (1) which links of the IIS pathway were involved in stress response and (2) what effect does the participation of the IIS pathway in stress response have on its ability to control the carbohydrate and lipid metabolism.
It was demonstrated by us earlier that in D. melanogaster females dFOXO translocates to the nucleus under heat stress (Gruntenko et al., 2016), and here we showed that this trans location is accompanied by a tendency to an increase of dfoxo expression (see Fig. 2). Our data also allow us to assume that dFOXO activation under stress results in dilp6 being inhibited as the decrease of dilp6 expression found in the control strain w 1118 is not observed in foxo BG01018 mutants (see Fig. 2).
dilp6 expression in the fat body was previously shown to suppress dilp2 and dilp5 expression in imago's brain and the secretion of DILP2 into hemolymph; dFOXO's influence on the expression of DILPs produced in median neurosecretory cells is inhibited by a simultaneous repression of DILP6 in the fat body via RNA interference (Bai et al., 2012). This allow us to suppose that a decrease in DILP6 activity under heat stress leads to an increase in level of DILPs expressed in median neurosecretory cells of the brain. Indeed, we were able to demonstrate earlier that DILP3 synthesis in these cells is increased in response to heat stress in wild type flies (An dreenkova et al., 2018), and in dilp6 41 larvae -under normal conditions (Andreenkova et al., 2017), which corresponds well with our assumption about a signal being transmitted from dFOXO to DILP3 through DILP6 under heat stress. Then, DILP3 appears to activate dInR, inhibiting the IIS pathway, which is confirmed by our data on the lack of a shift in dInR expression level under heat stress in flies with mutations of dilp6 and dfoxo genes as opposed to the shift in laboratory strain w 1118 , in which a decrease in dilp6 and an increase in dInR expression is shown to occur in response to heat stress (see Fig. 2).
System defects in the IIS pathway cause D. melanogaster to manifest a number of different phenotypes including those connected to metabolism, which usually involves an increase in organism's carbohydrates and lipids reserves (Mattila, Hie takangas, 2017). MurilloMaldonado et al. (2011) demon strated almost all viable combinations of mutations with partial loss of function or hypomorphism of IIS genes to have changes in carbohydrates and lipids levels. Slaidina et al. (2009) showed dilp6 knockdown to cause an increased level of triglycerides and glycogen in Drosophila larvae.
dFOXO controls the expression of insulin pathway genes and lipids content under stress in Drosophila These results correspond well with our data on the increased content of total lipids in females of dilp6 41 and foxo BG01018 strains (see Fig. 3, a), as well as with increased glucose and trehalose levels in dilp6 41 and foxo BG01018 mutants we previ ously demonstrated (Eremina et al., 2019).
Regarding regulation of feeding behaviour under heat stress it seems to occur independently from dilp6 and dfoxo genes as their mutations do not inhibit loss of appetite following stress (see Fig. 3, b).

Conclusion
Thus, we have shown that the disruption of dilp6 and dfoxo gene functions in Drosophila melanogaster (1) results in the feedback loop of the IIS pathway being disrupted under heat stress, (2) leads to an increase in total lipids content under normal conditions and impedes their decrease following heat stress, and (3) causes an increase in feeding intensity under normal conditions but does not impede its decrease following heat stress.